The overall objective(s) of the proposed research is to gain a fundamental understanding of the mechanism(s) involved in the temporal regulation of the expression of DNA repair genes. A thorough analysis of the regulatory mechanisms involved in the regulation of DNA repair processes will increase our understanding of cell cycle mediated gene regulation in eukaryotes and our knowledge of the strategies cell use to ensure self perpetuation without error. The information gained on understanding the regulation of the expression of DNA repair processes should provide insight into the relationship between DNA repair and genetic diseases in which predisposition to cancer is a phenotypic characteristic. The temporal modulation of DNA repair processes appears to be a common feature in mammalian cells of different origins. In addition, there appears to be a correlation between altered cell cycle control of DNA repair processes and DNA repair deficiency or hypersensitivity to damage. However, the exact mechanism of this modulation is unclear. Several questions remain unanswered: Is the modulation at the level of transcription or post transcriptional? Is control initiated at translation or post translational modification; Alternatively, it could be a combination of all of the above. It is clear though, that both human and CHO cells will provide good models for understanding the regulatory processes involved. As part of a continuing effort to understand the regulatory mechanism(s) involved in the temporal modulation of DNA repair processes as well as the molecular basis for the genetic defect in Bloom's syndrome and repair deficient CHO cell lines, it is proposed to examine the control of DNA repair processes at the molecular level. It is planned to look at control mechanisms from several different aspects; 1) To examine whether or not control is at the level of transcription. It is planned to quantitate mRNA levels of various DNA repair genes throughout the cell cycle using Northern Blot hybridization; 2) To clone and sequence the major apurinic/apyrimidinic (AP) endonuclease from Chinese hamster cells. It is planned to determine if there are any DNA sequence irregularities between the normal and the repair deficient AP endonuclease which could explain the observed difference in temporal modulation; 3) To construct a genomic library from CHO cells. The resulting library will be screened for uracil DNA glycosylase (UDG) and AP endonuclease genes to look for common regulatory regions which may explain the coordinate control of these gene products; 4) Student training is an integral component of the proposed research. Graduate students will be given the opportunity to be trained in modern molecular biology techniques while developing an appreciation for the rigors of scientific endeavor.
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